Structure-Guided Evaluation of Thiol-Based Fluorescent Probes for Selective Detection and Inhibition of...

Metallo-β-lactamases (MBLs)-Zn²⁺-dependent β-lactamases that compromise the efficacy of most β-lactam antibiotics, including carbapenems-are difficult to monitor rapidly with broadly applicable small-molecule probes due to the structural diversity among the MBLs. Fluorescent thiol-based probes that bind to the dinuclear Zn²⁺ active site would offer a mechanistically different approach for the selective detection and inhibition of MBLs compared to serine β-lactamases (SBLs); however, their performance across genetically divergent MBLs remains poorly defined. To clarify the structural determinants governing probe recognition across different MBLs, we examined the structure-activity relationships using a homologous series of dansyl-based thiol probes, DansylC_nSH (n = 2 and 4-6) toward BlaB, a class B MBL of Elizabethkingia meningoseptica, and compared their behavior with those of the clinically relevant MBLs imipenemase-1 (IMP-1) and Verona integron-encoded metallo-β-lactamase 2 (VIM-2). Consistent with the minimal differences in fluorescence enhancement among the series, BlaB inhibition exhibited only modest spacer-length dependence (IC₅₀ = 29-51 μM; apparent inhibition constants (K_i^app) = 3600-5910 nM). In contrast, IMP-1 and VIM-2 showed markedly stronger fluorescence emission and inhibitory potencies (IMP-1: IC₅₀ = 0.7-5.2 μM; K_i^app = 140-1100 nM; VIM-2: IC₅₀ = 1.5-2.1 μM; K_i^app = 286-370 nM), reflecting their distinct active-site architectures. Docking analyses further revealed distinct binding orientations that explain their photophysical behaviors. No fluorescence enhancement or inhibition was observed with the class A SBL Guiana extended-spectrum β-lactamase-3 (GES-3), confirming the high selectivity and specificity of the DansylC_nSH series. These findings allow identifying key structural determinants governing selective activation and inhibition of fluorescence from each MBL.